Sığır oositlerinin in vitro olgunlaştıılmasında hepes ve hormonların etkilei

Çalışmada, inek oositlerinin in vitro oyunlaştırılması için HEPES ile FSH, LH ve Estradiol 17-ß hormonlarının olgunlaşma oranlan üzerine etkilerinin incelenmesi amaçlanmıştır. Ovaryumlardaki 2-6 mm çaptaki folliküllerinden aspirasyonla kazanılan oositlerin olgunlaştırılmasmda Grup I. TCM 199 %10ECS + pyruvat (0,2 mM) + FSH (0,5 $mu$g/ml) + LH (5 $mu$g/ml) + Östrojen (1$mu$ m) Grup 2. TCM 199 +%IO ECS + pyruvat (0,2 mM) (kontrol); Grup 3. TCM + HEPES + % 10 ECS + pyruvat (0,2 mM) + FSH (0,5 $mu$m) + LH (5 $mu$g/ml) + Östrojen ( 1$mu$g/ml) ve Grup 4. TCM 199 + HEPES +%10 ECS + pyruvat (0,2 $mu$M) (kontrol) medyum kombinasyonları kullanılmıştır. Oositler 24 saat %5 $CO_2$ 38.5°C sıcaklık ve %95-100 nem bulunan ortamda inkübasyona bırakılmıştır. Olgunlaşan oositler kumulııslarından arındırılarak fikse edilmiş ve aselo-or-sein boyası ile boyanmıştır. Faz kontrast mikroskop altında x400 büyütmede germinal vezikül (GV), Metafaz l (M I) ve Metafaz II (M II) aşamaları belirlenmiştir. Veriler SPSS istatistik programında ki-kare yöntemiyle karşılaştırılmış ve önemliDk kontrolü gerçekleştirilmiştir. Birinci grupta 78, 2. grupta 82, 3. grupta 79 ve 4. grupta 79 adet olmak üzere toplam 318 adet oosit incelenmiştir. Oositlerden l. gruptaki 23 tanesinin (%29.49) M I aşamasına geliştiği 55 tanesinin (%70.51) ise M II aşamasına, 2. gruptaki oositlerden sırasıyla 2 (%2.44) tanesinin GV aşamasında kaldığı, 28 tanesinin (%34.15) M I ve diğer 52 (%63.41) tanesinin M II aşamasına, 3.gruptaki oositlerden 11 (%13.92) tanesinin M I aşamasına, 68 (%86.08) tanesinin M II aşamasına, 4. gruptaki oositlerden 18 tanesinin (%22.78) M I aşamasına, 61 (%77.22) tanesinin ise M II aşamasına ulaştığı belirlendi, istatistik analizi sonucunda gruplar arasında oluşan gelişim değerlerindeki farklar önemsiz bulundu ($x^2$ =3.879: (P

The effects of HEPES and hormones on in vitro maturation of cattle oocytes

The aim of the study was to investigate the effect of HEPES, FSH, LH and estradiol 17-ß hormones on in vitro maturation of cattle oocytes. Oocytes were recovered from ovaries of slaughtered cows by aspiration of 2-6 mm diameter follicles. Recovered oocytes were matured in four different combinations of media as 4 groups. In the first group TCM 199 + 10% ECS + pyruvate (0,2 mM) + FSH (0,5 $mu$g/ml) +LH (5 $mu$g/ml) + Estradiol 17-ß (1 $mu$g/ml). in the second group TCM 199 + 10% ECS + pyruvate (0,2 mM) (as control), in the third group TCM 199 + HEPES + 10% ECS + pyruvate (0,2 mM) + FSH (0,5 $mu$g/ml) + LH (5 $mu$g/ml) + Estradiol 17-ß (1 $mu$g/ml), İn the fourth group TCM 199 + HEPES + 10% ECS + pyruvate (0,2 mM) (as control) combinations were used as maturation media. Oocytes were incubated in 5% $CO_2$ in 95-100% humidified atmosphere at 38,5°C for 24 hours. Matured oocytes were denuded from cumulus cells and fixed with acetic aside/ethanol (1/3 v/v) and then stained by aceto orcein. Germinal vesicle (GV), metaphase I (M I), and metaphase II (M II) stages were determined under a phase contrast microscope at x400 magnification. The data were compared by chi-square method at SPSS statistics program and significance analyses were done. To-tal 318 oocytes were examined in four groups, 78 oocytes in the first group, 83 oocytes in the second group, 79 oocytes in the third and 79 oocytes were used in the fourth groups. In the first group 23 oocytes (29,49%) matured to M I stage, and 55 (70,51%) oocytes matured to M II stage, 2 oocytes (2,44%) of the second gro-ups stayed at GV stage, 28 oocytes (34,15%) matured to MI and 68 oocytes (86,08%) matured to M II stage, 11 oocytes ( 13,92%) of the third groups oocytes matured to M I stage and 68 oocytes (86,08%) matured to M II stage, 18 oocytes (22,78%) of the fourth group oocytes matured to M I stage and 61 oocytes (77,22%) matured to M II stage. By the statistical analysis, differences among developmental values were not important ( $X^2 $= 3,879: P< 0,275). In conclusion, considering the highness of numeric values and literature data, TCM + HEPES + %10 ECS + pyruvat (0,2 mM) + FSH (0,5 $mu$g/ml) + LH (5 $mu$g/ml) + Estradiol 17-ß (l ug/ml) combination was decided to be suitable for using in maturation procedure of the cattle oocytes in vitro.

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